Diagnostic composition and method for the detection of nitrate reduction

ABSTRACT

A diagnostic composition and method for detecting the reduction of nitrate to nitrite by micro-organisms which comprises a carrier material impregnated in specific zones with a nitratecontaining medium, a barrier composition, and two stable reagent solutions. In use, a suspension of the culture to be tested is incubated in the presence of the nitrate medium and the reduction of nitrate to nitrite is indicated by the formation of color in the reagent zones.

ilnited States Patent Kronish et al.

[ Feb. 29, 1972 DIAGNOSTIC COMPOSITION AND METHOD FOR THE DETECTION OF NETRATE REDUCTION lnventors: Donald P. Kronish, Rockaway; William D.

Young, Jr., Montclair, both of NJ.

Warner-Lambert Company, Morris Plains, NJ.

Filed: June 24, 1969 Appl. No.: 836,089

Assignee:

US. Cl. ..l95/l03.5 R, 23/253 TP, 195/100 Int. Cl. ..C12k 1/06 Field ofSearch ..195/l03.5;260/l92,196

References Cited UNITED STATES PATENTS 9/1967 Evans et a1 ..l95/103.5 Watson et a1. ..l95/103.5 X

3,547,780 12/1970 Finnerty ..23/253 TPX OTHER PUBLICATIONS I Welcher, Organic Analyt. Reagents Vol. [V pp. 574- 583 (1948).

Primary Examiner-Lionel M. Shapiro Assistant Examiner-Max D. Hensley Attorney-Albert H. Graddis, Henry E. Millson, .lr., Frank S. Chow, Neil D. Edwards and Anne M. Kelly [57] ABSTRACT A diagnostic composition and method for detecting the reduction of nitrate to nitrite by micro-organisms which comprises a carrier material impregnated in specific zones with a nitratecontaining medium, a barrier composition, and two stable re- 1 1 Claims, 1 Drawing Figure RAIENIEDfEazswsn AREA l l4mm ITAREAIZT AREA 5 l4 mm AREA 4 l6 mm AREAS l6.5 mm

AREA 6 ZONE ZONE

ZONE

I REVERSE SIDE zone zone zone

1 v INVENTOR.

BY WILLIAM 0. vouue, JR.-

BACKGROUND OF THE INVENTION The taxonomic value of the nitrate reduction test for the identification and differentiation of micro-organisms which are pathogenic to humans is well known. The Enteriobacteriaceae family react positively to the nitrate reduction test. Certain bacteria reduce nitrate to nitrite only while others continue the reduction and transform nitrite compounds into free nitrogen or ammonia.

In spite of its value, the utility of the nitrate reduction test in the clinical laboratory has been limited, due primarily to the fact that the time required to obtain results is too long to be of real value. Furthermore, known test methods require the careful preparation of necessary reagents immediately prior to use.

7 DESCRIPTION OF PRIOR ART Procedures for the determination of the ability of bacteria to reduce nitrate to nitrite are known. In the classical test, a

nutrient medium containing potassium nitrate (nitrite free) is inoculated with a pure culture of the strain under examination and incubated at 37 C. for 24 hours. The medium is tested for the presence of nitrites by adding 0.1 ml. of a mixture of test reagents prepared from 0.08% w/v sulfanilic acid in 5 N acetic acid; and 0.5% w/v a-naphthylamine I-aminonaphthalene) in 5 N acetic acid. These reagents are mixed together immediately before use. The development of a distinct pink or red color in the test medium, after the addition of the mixture of reagents, indicates the presence of nitrite produced from the original nitrate (Identification of Enterobacteriaceae, P. R. Edwards and W. H. Ewing, Burgess Pub. Co., 1962, printed 1964).

Another work (Quicker Bacteriological Results, R. H. Weaver, Am. J. Med. Tech., 14-26, 1954) indicates that an incubation period of 15 minutes is possible using a medium containing peptone, beef extract, and potassium nitrate; however, the usual nitrite reagents must still be added to obtain color development as an indication that the reduction of nitrate to nitrite has taken place.

Thus, it can be seen that while some progress has been made, prior art methods still involve painstaking preparation of critical test reagents which can only be mixed immediately prior to use. The prior art procedures all have the disadvantage that they require a considerable amount of knowledge and a high degree of skill in order to obtain reliable, reproducible test results.

OBJECTS OF THE PRESENT INVENTION It is the object of this invention to provide an improved diagnostic composition for the rapid identification and differentiation of micro-organisms which reduce nitrate to nitrite.

Another object of this invention is to provide a diagnostic composition in which all the required nutrients and reagents for said differentiation test are provided in a premeasured, stable, easy-to-use form.

It is still another object of this invention to provide a diagnostic composition in which all the required nutrients and test reagents are incorporated on a single-carrier support material.

It is a further object of this invention to provide a rapid, sensitive and accurate test procedure which utilizes the diagnostic composition of this invention but which requires only about 2 hours for completion.

Other objects will appear from the following detailed description.

SUMMARY OF THE INVENTION A rapid, stable diagnostic composition and method for the identification and differentiation of micro-organisms which reduce nitrate to nitrite is provided in the form of an impregnated carrier material. The use of the composition of this invention makes it possible to enable one to differentiate between micro-organisms, which do or do not reduce nitrate in as little as about 2 hours since it is sensitive to the detection of as little as l microgram of nitrite. The diagnostic composition provided is stable for at least 12 months at room temperature.

DESCRIPTION OF THE DRAWING The accompanying drawing is a diagrammatic representation of a bibulous test strip of this invention impregnated with Zone A nitrate medium in area l,'Zone B hydrophobic barrier in area 2, Zone C reagent on one side of area 3, Zone D reagent on the reverse side of area 3, Zone B, hydrophobic barrier in area 4, and Zone E Dye in area 6; area 5 is untreated.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The diagnostic composition of this invention is prepared by impregnating certain specified areas of a bibulous carrier material with a solution of a nitrate-containing medium (hereinafter designated Zone A Nitrate Medium) and stable reagent solutions (hereinafter designated Zone C and Zone D Reagents), wherein the nitrate medium is separated from the reagent zone area by a hydrophobic barrier composition (hereinafter designated Zone B Hydrophobic Barrier). An additional barrier zone, an untreated area and a dyed identification zone are optionally present in the preferred diagnostic composition.

The impregnated bibulous carrier material is cut into individual strips containing sufficient quantities of all the ingredients necessary for the identification and differentiation of micro-organisms by their ability to reduce nitrate to nitrite.

The Zone A nitrate-containing medium can be any composition containing a nontoxic nitrate salt alone, or, preferably in combination with a nutrient medium such as peptone. A preferred medium for example, is prepared by forming an aqueous solution of beef extract, peptone and potassium nitrate. A particularly preferred medium of this type, marketed as BACTO-NITRATE BROTH (dehydrated) by Difco Laboratories, Detroit, Michigan, contains these components in a ratio of 3 g. of beef extract, to 5 g. of peptone and l g. of potassium nitrate. It has been found that a [00 ml. distilled water solution of from about 10 to 27 g. of dehydrated BACTO-NITRATE BROTH is quite suitable for use in the preparation of the diagnostic composition of this invention.

As the Zone B hydrophobic barrier, the composition used is one which will prevent the premature leaching of the culture during incubation upward into the reagent zone.

The barrier composition must, of course, be chemically and biologically inert in this test system. Any substance which will form a waterproof barrier of this type may be sued. Suitable materials include waxes, lacquers, and plastics, particularly that colorless polymerized methyl methacrylate coating composition marketed by Krylon, Inc., Norristown, Pa. under the trade name KRYLON 150 CRYSTAL CLEAR. The KRYLON material is particularly preferred. It is supplied in a toluene vehicle and may be diluted for ease of application with additional toluene or other diluents, such as ethyl, methyl, or propyl alcohol USP.

It has been found that a barrier solution prepared from about 75 to ml. of KRYLON and to which is added 0 to 25 ml. of diluent is suitable.

Two reagent zones are provided on the diagnostic test product of this invention. Zone C Reagent contains an aminosubstituted-naphthalene sulfonic acid and an alkali metal salt of sulfanilic acid or, alternately, sulfadiazine or an alkali metal salt of sulfathiazole while Zone D Reagent contains a crystalline acid such as oxalic, malonic or citric acid. The amino-substituted-naphthalene sulfonic acid may, for example, be 5- amino-Z-naphthalene sulfonic acid, 8-amino-2naphthalene sulfonic acid or S-amino-l-naphthalene sulfonic acid, with the S-amino-l-naphthalene sulfonic acid preferred among the three acids. As the salt of sulfanilic acid, the sodium salt is preferred. The two reagent impregnated zones are positioned adjacent to each other on the individual diagnostic test product, either in a side-by-side relationship on the same side of the carrier material or, preferably, positioned back-to-back with one of the reagent solutions impregnated on one side and the other reagent on the other side of the same area of the carrier material. The two reagent solutions are applied to the bibulous carrier material separately, allowing drying between applications to prevent premature mixing of the reagents prior to wetting during the performance of the test.

Zone C Reagent solution is prepared from a 100 ml. distilled water solution of:

1. from about 0.1 to 4 g., preferably about 0.2 to 2 g. of an amino-substituted-naphthalene sulfonic acid; and

2. from about 0.16 to 7 g., preferably about 0.3 to 4 g. of an alkali metal salt of sulfanilic acid.

The pH of the final solution of Zone C Reagent is adjusted to from about 7 to 12, preferably from about 9.8 to 10, with a suitable pH modifying agent which will not interfere with the diagnostic test, suchas a sodium, potassium,-or ammonium hydroxide; preferably an 0.1 N sodium hydroxide solution is used. In the preferred embodiment of the invention, an interim pH adjustment is conveniently made on an aqueous solution of the amino-substituted-naphthalene sulfonic acid,

using a sufficient amount of a pH modifying agent (preferably 1 N sodium hydroxide) to adjust this solution to a pH of from about 7 to 12, preferably about 9.5 to 10. Thereafter, the sulfanilic acid salt is added and the solution is brought to a volume of 100 ml. and the final pH adjustment is made.

Zone D Reagent solution is prepared from a solution of from about 20 to 65 g., and preferably about 35 to 60 g. in 100 ml. of distilled water of at least one of the crystalline acids such as oxalic, malonic or citric acid. The Zone D Reagent solution described is preferably applied to the reverse side of that portion of the reagent zone on the carrier material which carries the Zone C Reagent solution.

Each of the above Zone A, C and D solutions are applied to the bibulous material so as to provide on each individual diagnostic test product, the following amounts ofingredients:

Broad Range Narrow Range Optionally, one end of the impregnated test strip may contain additional, zones to prevent the contamination of the nitrate medium and reagent zones through handling. For example, there may be present adjacent to the outermost reagent zone at one end of the strip, an additional barrier zone hereinafter designated Zone B hydrophobic barrier, further, a dyed identification zone hereinafter designated Zone E Dye may be present contiguous to barrier Zone B Barrier, or separated from this second barrier zone by an untreated area.

The previously described Zone l3 hydrophobic barrier composition is also suitable for use as the Zone B hydrophobic barrier.

Any suitable dye which will color the bibulous material sufficiently to distinguish the end of the diagnostic test strip which is to be handled from the colorless reagent zones which are to be inserted into the culture under investigation may be used. About 0.025 to 0.3 g. of a dye, dissolved in a suitable solvent and adjusted to a volume of 100 ml. has been found to provide a suitable identification Zone E solution. The preferred dye solution is prepared from brilliant green, a biological stain (Matheson, Coleman and Bell) which is soluble in water. However, many other dye solutions would be equally effective, for instance Methyl Green (National Aniline).

The bibulous materials suitable as the carrier for the diagnostic composition of this invention are those materials which, by means of capillary action, are able to hold liquid. Such materials include filter paper, felt, porous ceramic strips, woven or matted glass fiber and the like. A particularly preferred pater is Eaton-Dikeman No. 623 (70 lbs).

In the preferred embodiment of the invention, a single-diagnostic composition, as pictured in the accompanying drawing, contains:

Area 1:

Zone A: about 2.8 mg. of nitrate broth prepared from beef extract, peptone and potassium nitrate; Area 2:

Zone 8,: saturated with a solution of about ml. of a methyl methacrylate resin coating composition and about 15 ml. of ethyl alcohol;

Area 3:

Zone C: about 0.05 mg. of 5amino-l-naphthalene sulfonic acid and about 0.08 mg. of the sodium salt of sulfanilic acid; Area 3:-

Zone D: about 3 mg. of citric acid;

Area 4:

Zone 8,: totally saturated with the Zone B solution; Area 5: I

Untreated Area Area 6:

Zone E: visibly colored by the application of a ml. aqueous solution of 0.1 g. of brilliant green. The diagnostic composition of this invention is stable for at least 12 months at 4 C. and at room temperature.

To determine the reduction of nitrate to nitrate by a particular micro-organism, a loopful of the culture to be tested is suspended in 0.3 ml. of saline in a l3Xl00 mm. or similar size test tube. The diagnostic composition test strip of this invention is inserted in the tube in such a manner that the Zone A nitrate medium is immersed in the test suspension. The tube is incubated at from'35 to 37 for about 1 A to 2 hours. The tube is then tipped to wet the reagent zone areas and the development of a pink to red color in the reagent zone in 30 seconds to 10 minutes indicates a positive result, i.e., that nitrate has been reduced to nitrite. No color change or a light buff color indicates a negative test.

The diagnostic composition of this invention will detect the presence of 1 microgram of nitrite ion present in 0.3 ml. suspension ofa culture to be tested.

The diagnostic composition and procedure of this invention was compared with the results obtained using classical method of Edwards and Ewing (Identification of Enterobacteriaceae, P. R. Edwards and W. H. Ewing, Burgess Pub. Co. 1962, printed 1964). A total of 107 organisms were treated. Results are listed in Table 1 below. 106 of the cultures gave the same reaction with both procedures, a correlation of better than 99 1 Strip-broth weak.

The following examples are included to further illustrate this invention:

EXAMPLE 1 A. Preparation of Zone A Nitrate Medium Solution To 60 ml. of distilled water, add g. of Bacto-Nitrate Broth, dehydrated (Difco). Mix well, and adjust volume to 100 ml. with distilled water. Heat to 100 C., and boil briefly to obtain a clear solution, then cool to C. Use for application to test strip within 2 hours of preparation.

B. Preparation of Zone B, and Zone B Hydrophobic Barrier Composition Dilute 85 ml. of Krylon 150 Crystal Clear with 15 ml. of 95 percent ethanol, USP.

C. Preparation of the Zone C reagent Solution, Amino Substituted Naphthalene Sulfonic Acid and Sulfanilic Acid Salt To 50 ml. of distilled water, add 1 g. of S-amino-lnaphthalene sulfonic acid. Slowly add, with mixing, 1 N sodium hydroxide solution until a stable pH of 9.5-10 is obtained. Add 1.6 g. of sulfanilic acid, sodium salt, mix to dissolve and adjust to a pH of 9.8l0 with 0.1 N NaOH solution. Adjust volume to 100 ml. with distilled water and mix.

D. Preparation of the Zone D Reagent Solution, Citric Acid To 50 g. citric acid, add sufficient distilled water to obtain a final volume of 100 ml. Mix and warm as necessary to dissolve.

E. Preparation of Zone E, Dye Solution Dissolve 0.1 g. brilliant green, biological stain (Matheson, Coleman & Bell) in distilled water, adjust volume to 100 ml. with distilled water and mix.

EXAMPLE 2 Application of Diagnostic Solutions to Bibulous Material in producing the strips, a continuous sheet of Eaton- Dikeman Filter Paper No. 623 (70 lbs.), 83 mm. in width, is employed. The paper is divided into six separate areas as shown in the accompanying drawing.

Apply the Zone B hydrophobic Barrier Solution of Example 1 to areas 2 and 4 in amounts sufficient to saturate the paper and allow to dry. Apply the Zone A Nitrate Medium Solution of Example 1 to area 1, once to each side of the paper and allow to dry. Apply the Zone E Dye Solution of Example 1 to area 6 once to each side of the paper and allow to dry. Apply Zone C Reagent Solution of Example 1 to one side of area 3, limiting the amount applied so that no more than one-half the thickness of the paper is wet. Allow to dry. Apply Zone D Reagent Solution of Example 1 to the reverse side of area 3 so that no more than one-half the thickness of the paper is wet. The reagent solutions must be applied carefully in order that Zone C reagent solution does not mix with Zone D reagent solution. After all areas of the paper have dried thoroughly, cut into one-quarter inch (6.3 mm.) strips, each containing the 6 areas impregnated with solutions herein described. Generally, the nitrate broth is applied to the paper at a rate of 0.70 ml. for each cm. of length with the sulfanilic acid and the S-amino-naphthalene sulfonic acid being applied at a rate of 0.25 ml. and the citric acid solution being applied at a rate of0.3 ml. per 30 cm.

EXAMPLE 3 Use of the Diagnostic Composition Method of Use l. Suspend a loopful of culture from an agar medium in 0.3

ml. of saline in a 13X 100 mm. or similar size tube.

2. Add the diagnostic composition test strip prepared according to Examples 1 and 2 to the suspension so that the zone opposite the green marker zone is immersed.

3. incubate the tube in a water bath for 2 hours at -37 4. Tip the tube to wet the reagent zone. Development of a pink to red color in the reagent zones in 30 seconds to 10 minutes indicates a positive test. No color change or a light buff color indicates a negative test.

EXAMPLE 4 A. Preparation of Zone A Nitrate Medium Solution.

To 60 ml. of distilled water, add 27 g. of Bacto-Nitrate Broth, dehydrated (Difco). Mix well, and adjust volume to 100 ml. with distilled water. Heat to 100 C. and boil briefly to obtain a clear solution, then cool to 25 C. Use within 2 hours of preparation.

B. Preparation of Zone B, and Zone B Hydrophobic Barrier Solution.

Dilute ml. of Krylon 150 Crystal Clear with 1 5 ml. of toluene.

C. Preparation of the Zone C Reagent Solution, Amino-Substituted Naphthalene Sulfonic Acid and Sulfanilic Acid Salt.

To 50 ml. of distilled water, add 1 g. of 8-amino-2- naphthalene sulfonic acid. Slowly add, with mixing, 1.0 N sodium hydroxide solution until a stable pH of -10 is obtained. Add 1.6 g. of sulfathiazole, sodium slat, mix to dissolve and adjust to a pH of 9.8-10 with 0.1 N NaOH solution. Adjust volume to ml. with distilled water and mix.

D. Preparation of the Zone D Reagent Solution, Malonic Acid.

To 50 g. malonic acid, add sufficient distilled water to obtain a final volume of 100 ml. Mix and warm as necessary to dissolve. -amino- 1 -naphthalene-sulfonic E. Preparation of Zone E, Dye Solution.

Dissolve 0.1 g. Methyl Green (National Aniline) in distilled water, adjust volume to 100 ml. with distilled water and mix.

EXAMPLE 5 Apply the Zone A, B, C, D and E solutions, prepared according to Example 4 to the bibulous material carrier as described in Example 2.

EXAMPLE 6 Follow the procedure of Example 3, using the diagnostic composition test strip prepared according to Examples 4 and 5.

We claim: 1. A diagnostic composition for the detection of nitrite, which comprises a bibulous material containing at least four impregnated zones, wherein:

A. Zone A contains a nitrate-containing medium; B. Zone B contains a dried inert hydrophobic barrier composition, separating Zone A from all succeeding impregnated zones; C. Zone C contains (1) an alkali metal salt of an amino-substitutednaphthalene sulfonic acid of the group consisting of 5- amino-2-naphthalene-sulfonic acid, 8-amino-2- naphthalene-sulfonic acid, and S-aminol naphthalene-sulfonic acid, and

(2) a reagent of the group consisting of an alkali metal salt of sulfanilic acid, sulfadiazine and an alkali. metal salt of sulfathiazole; and

D. Zone D contains at least one crystalline acid of the group consisting of citric acid, oxalic acid and malonic acid; said zones being arranged in an order which will promote the development of color in the reagent Zones C and D to detect any reduction of the nitrate present to nitrite by micro-organisms incubated in the presence of said nitrate-containing medium Zone A.

2. A diagnostic composition for the detection of nitrite, which comprises a bibulous material containing at least four impregnated zones, wherein:

A. Zone A contains from about 1.4 to about 4.6 mg. of a nitrate-containing nutrient medium;

B. Zone B contains a dried inert hydrophobic barrier composition, separating Zone A from all succeeding impregnated zones;

C. Zone C contains 1 from about 0.005 to about 0.2 mg. of an alkali metal salt of an amino-substituted-naphthalene sulfonic acid of the group consisting of 5-amino-2-naphthalene-sulfonic acid, 8-amino-2-naphthalene-sulfonic acid, and S-amino- 1 -naphthalene-sulfonic acid, and

(2) from about 0.008 to about 0.36 mg. of a reagent of the group consisting of an alkali metal salt of sulfanilic acid, sulfadiazine, and an alkali metal salt of sulfathiazole; and

D. Zone D contains from about 1 to about 4.5 mg. of at least one crystalline acid of the group consisting of citric acid, oxalic acid and malonic acid; said zones being arranged in an order which will promote the development of color in the reagent Zones C and D to detect any reduction of the nitrate present to nitrite by micro-organisms incubated in the presence of said nitrate-containing nutrient medium Zone A.

3. A diagnostic composition according to claim 2, wherein an additional barrier Zone B, containing a dried. inert hydrophobic barrier composition, is positioned contiguous to the outermost reagent impregnated zone.

4. A diagnostic composition according to claim 3, wherein an additional Zone E, containing a dried identifying dye on one end of the diagnostic composition test strip is positioned at the end opposite the nutrient medium Zone A.

5. A diagnostic composition for the detection of nitrite, which comprises a bibulous material containing at least four reagentimpregnated zones, wherein:

A. Zone A contains from about 2 to about 4 mg. ofa nitratecontaining nutrient medium;

B. Zone B contains a sufficient amount of a dried inert hydrophobic barrier composition to saturate Zone B, and separate Zone A from all succeeding impregnated zones, said inert hydrophobic barrier composition comprising an acrylic coating composition;

C. Zone C contains 1. from about 0.01 to about 0.1 mg. of an alkali metal salt of an amino-substituted-naphthalene sulfonic acid of the group consisting of 5-amino-2-naphthalene-sulfonic acid, 8-amino-2-naphthalene-sulfonic acid, and S-amino-l-naphthalene-sulfonic acid, and

2. from about 0.015 to about 0.2 mg. ofa reagent of the group consisting of alkali metal salt of sulfanilic acid, sulfadiazine, and an alkali metal salt of sulfathiazole; and

D. Zone D contains from about 2 to about 4 mg. of at least one crystalline acid of the group consisting of citric acid, oxalic acid, and malonic acid; said zones being arranged in an order which will promote the development of color in the reagent Zones C and D to detect any reduction of the nitrate present to nitrite by micro-organisms incubated in the presence of said nitrate-containing nutrient medium Zone A.

6. A diagnostic composition according to claim 5, wherein an additional inert, hydrophobic barrier Zone B ispositioned contiguous to the outermost reagent impregnated zone.

7. A diagnostic composition according to claim 6, wherein an additional Zone E, positioned on one end of the diagnostic composition, contains a sufficient amount of a dried identifying dye to color Zone E, said additional Zone E being positioned at the end opposite the nitrate medium Zone A.

8. A diagnostic composition for the detection of nitrite, which comprises a bibulous material containing at least four reagent impregnated zones, wherein:

A. Zone A contains about 2.8 mg. of a nutrient medium prepared from about 3 mg. of beef extract, about 5 mg. of peptone and about 1 mg. of potassium nitrate;

B. Zone B contains a sufficient amount of a dried inert hydrophobic barrier composition to saturate Zone B, said hydrophobic barrier composition comprising a methyl methacrylate coating composition;

C. Zone C contains 1. about 0.05 mg. of the sodium salt of 5-amino-lnaphthalene sulfonic acid, and 2. about 0.08 mg. ,of the sodium salt of sulfanilic acid; and

D. Zone D contains about 3 mg. of citric acid; wherein the zones are positioned on the bibulous material in the sequence A, B, C with Zone D applied to the bibulous material on the reverse side of Zone 0 and wherein Zone A 15 contiguous only to Zone B; Zone B 15 contiguous only to Zone A and Zone C on one side of the bibulous material and contiguous only to Zone A and Zone D on the reverse side of the bibulous material; and Zone C and Zone D are positioned in a back-to-back relationship to each other and are each contiguous only to Zone B and to each other.

9. A diagnostic composition according to claim 8, wherein an additional inert, hydrophobic barrier Zone B is positioned contiguous to the outermost reagent impregnated zone.

10. A diagnostic composition according to claim 9, wherein an additional Zone E positioned on one end of the diagnostic composition test strip, contains a sufficient amount of a dried identifying dye to color Zone E, said additional Zone E being positioned at the end opposite the nitrate medium Zone A.

11. A process for the identification and differentiation of micro-organisms which reduce nitrate to nitrite which comprises:

A. immersing Zone A of the diagnostic composition of claim 1 into a test tube containing a saline suspension of the culture to be tested;

B. Incubating the test tube for about 1.5 to about 2 hours at about 35 C. to about 37 C;

C. Tipping the test tube to wet Reagent Zones C and D; and

D. Allowing from 30 seconds to 10 minutes to elapse for color to develop as a positive indication that the nitrate present has been reduced to nitrite by the micro-organism. 

2. A diagnostic composition for the detection of nitrite, which comprises a bibulous material containing at least four impregnated zones, wherein: A. Zone A contains from about 1.4 to about 4.6 mg. of a nitrate-containing nutrient medium; B. Zone B contains a dried inert hydrophobic barrier composition, separating Zone A from all succeeding impregnated zones; C. Zone C contains 1 from about 0.005 to about 0.2 mg. of an alkali metal salt of an amino-substituted-naphthalene sulfonic acid of the group consisting of 5-amino-2-naphthalene-sulfonic acid, 8-amino-2-naphthalene-sulfonic acid, and 5-amino-1-naphthalene-sulfonic acid, and (2) from about 0.008 to about 0.36 mg. of a reagent of the group consisting of an alkali metal salt of sulfanilic acid, sulfadiazine, and an alkali metal salt of sulfathiazole; and D. Zone D contains from about 1 to about 4.5 mg. of at least one crystalline acid of the group consisting of citric acid, oxalic acid and malonic acid; said zones being arranged in an order which will promote the development of color in the reagent Zones C and D to detect any reduction of the nitrate present to nitrite by micro-organisms incubated in the presence of said nitrate-containing nutrient medium Zone A.
 2. from about 0.015 to about 0.2 mg. of a reagent of the group consisting of alkali metal salt of sulfanilic acid, sulfadiazine, and an alkali metal salt of sulfathiazole; and D. Zone D contains from about 2 to about 4 mg. of at least one crystalline acid of the group consisting of citric acid, oxalic acid, and malonic acid; said zones being arranged in an order which will promote the development of color in the reagent Zones C and D to detect any reduction of the nitrate present to nitrite by micro-organisms incubated in the presence of said nitrate-containing nutrient medium Zone A.
 2. about 0.08 mg. of the sodium salt of sulfanilic acid; and D. Zone D contains about 3 mg. of citric acid; wherein the zones are positioned on the bibulous material in the sequence A, B, C with Zone D applied to the bibulous material on the reverse side of Zone C; and wherein Zone A is contiguous only to Zone B; Zone B is contiguous only to Zone A and Zone C on one side of the bibulous material and contiguous only to Zone A and Zone D on the reverse side of the bibulous material; and Zone C and Zone D are positioned in a back-to-back relationship to each other and are each contiguous only to Zone B and to each other.
 3. A diagnostic composition according to claim 2, wherein an additional barrier Zone B, containing a dried inert hydrophobic barrier composition, is positioned contiguous to the outermost reagent impregnated zone.
 4. A diagnostic composition according to claim 3, wherein an additional Zone E, containing a dried identifying dye on one end of the diagnostic composition test strip is positioned at the end opposite the nutrient medium Zone A.
 5. A diagnostic composition for the detection of nitrite, which comprises a bibulous material containing at least four reagent impregnated zones, wherein: A. Zone A contains from about 2 to about 4 mg. of a nitrate-containing nutrient medium; B. Zone B contains a sufficient amount of a dried inert hydrophobic barrier composition to saturate Zone B, and separate Zone A from all succeeding impregnated zones, said inert hydrophobic barrier composition comprising an acrylic coating composition; C. Zone C contains
 6. A diagnostic composition according to claim 5, wherein an additional inert, hydrophobic barrier Zone B is positioned contiguous to the outermost reagent impregnated zone.
 7. A diagnostic composition according to claim 6, wherein an additional Zone E, positioned on one end of the diagnostic composition, contains a sufficient amount of a dried identifying dye to color Zone E, said additional Zone E being positioned at the end opposite the nitrate medium Zone A.
 8. A diagnostic composition for the detection of nitrite, which comprises a bibulous material containing at least four reagent impregnated zones, wherein: A. Zone A contains about 2.8 mg. of a nutrient medium prepared from about 3 mg. of beef extract, about 5 mg. of peptone and about 1 mg. of potassium nitrate; B. Zone B contains a sufficient amount of a dried inert hydrophobic barrier composition to saturate Zone B, said hydrophobic barrier composition comprising a methyl methacrylate coating composition; C. Zone C contains
 9. A diagnostic composition according to claim 8, wherein an additional inert, hydrophobic barrier Zone B is positioned contiguous to the outermost reagent impregnated zone.
 10. A diagnostic composition according to claim 9, wherein an additional Zone E positioned on one end of the diagnostic composition test strip, contains a sufficient amount of a dried identifying dye to color Zone E, said additional Zone E being positioned at the end opposite the nitrate medium Zone A.
 11. A process for the identification and differentiation of micro-organisms which reduce nitrate to nitrite which comprises: A. Immersing Zone A of the diagnostic composition of claim 1 into a test tube containing a saline suspension of the culture to be tested; B. Incubating the test tube for about 1.5 to about 2 hours at about 35* C. to about 37* C; C. Tipping the test tube to wet Reagent Zones C and D; and D. Allowing from 30 seconds to 10 minutes to elapse for color to develop as a positive indication that the nitrate present has been reduced to nitrite by the micro-organism. 